The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and the aqueous compartment termed the periplasm that is located between the membranes. The long term goal of this grant has always been to understand the mechanisms of envelope biogenesis in molecular terms using Escherichia coli as a model system. Much of this proposal concerns OM biogenesis. All of the components of the OM, phospholipids (PL), lipopolysaccharide (LPS) lipoproteins (LP), and 2-barrel proteins (OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified most, if not all, of the essential proteins required to transport LPS and OMPs across the periplasm and assemble these molecules in the OM. To address the function of these proteins, we propose several strategies that will enable us to identify mutations that compromise the function of the components of both the LPS and the OMP assembly machines. In addition, we are developing a technique that combines Two-Photon and Total-Internal Reflection Fluorescent (TIRF) microscopy to probe the spatial localization of, and the interactions between these assembly machines. These studies will provide important information regarding the structure, function, and the component interactions within, and between, these assembly machines. Mechanistic hypotheses generated will be tested biochemically in an established, productive collaboration with the lab of Dan Kahne at Harvard. We have discovered an ABC transporter that functions with an OM LP in retrograde transport of PL from the OM to the IM. We will address the function of this transporter, and try to exploit its properties to identify components of the elusive anterograde PL transporter(s). Our interest in envelope stress responses has led to the discovery of two novel proteins. One of these appears to function as a global regulator of all the envelope stress responses. The other functions in a quality control mechanism that operates at the level of SecY and shows remarkable similarities to ER stress-induced apoptosis in eukaryotes. Experiments planned will address the functions of these proteins and what controls their activities. Contrary to current views, we have strong evidence for surface-exposed LPs in E. coli. We will address the mechanism that directs these molecules to the cell surface. Multi-drug resistant Gram-negative bacteria are a growing concern. The components that we have identified that are required for OM biogenesis are attractive drug targets. PUBLIC HEALTH RELEVANCE: The outer membrane of Gram-negative bacteria serves as a barrier to protect these organisms form toxic compounds, including antibiotics. We have identified two protein complexes in the outer membrane that function to assemble this essential cellular organelle, and we wish to understand how these assembly machines work in molecular detail. Such knowledge would facilitate the development of new antibiotics that would combat the growing problem of antibiotic resistance in this important class of pathogens.